EXERCISE NO. 4Atomic Absorption Spectrometry

CHEM 137.1 2LGroup # 2ROSALES, Abigail Jane C.BRITANIA, Stephanie H.PETERE, Mary Rose L.SUERTE, Clark T.

Date performed: September 11 & 18, 2015Date submitted: October 02, 2015

Submitted to: Sir Lloyd M. Lapoot

ABSTRACTAtomic absorption/ emission spectroscopy is governed by the absorption/emission of light to measure the concentration of gas-phase atoms especially metals. Using the working curve obtained from the standards and the application of Beer-Lambert’s Law, concentration of metals can be determined. The metals Ca2+ and K+ in Bear Brand milk powder and Pb2+ in a water sample were studied. Results showed that the obtained concentrations of the metals from the milk sample were 555.23 ppm Ca2+ and 183.60 ppm K+ while 4.73 ppm Pb2+ using external calibration and4.34 ppm Pb2+ using standard addition method from the water sample. Application of releasing agent, La, and suppressant, Cs, which limits the activity of a certain compound, was added to Ca2+ and K+ samples, respectively.

I. INTRODUCTION Atomic absorption spectrometry is used for the determination of the concentration and presence of a metal in the sample. Metals absorb ultraviolet light when they are excited and for each metal there is a characteristic wavelength that can be absorbed. AAS measures the change in intensity. Intensity is reduced when a metal is present and absorbs some light. The change in intensity can be read in the computer as the absorbance. Spectrophotometry, on the other hand, is the study of the absorption of light by an analytical sample.According to Skoog (2004), spectroscopic determination of atomic species can only be performed on a gaseous medium in which the individual atoms (or sometimes, elementary ions) are well separated from one another. Atomization is the first and the most critical step in all atomic spectroscopic procedures wherein the sample is volatilized and decomposed in such a way as to produce an atomic gas. The most widely used method to atomize the sample for atomic spectroscopic studies is flame atomization. The sample produced atomic absorption, emission and fluorescence spectra. Atomic absorption spectroscopy (AAS) measures the radiation absorbed by the atom in ground state when it is

excited to a higher energy level while atomic emission spectroscopy (AES) is the opposite which involves the quantification of discrete radiation that is emitted by excited atoms upon returning to the ground state. Atomic fluorescence spectroscopy (AFS), on the other hand, quantifies the amount of energy emitted by atoms that have been excited via radiation from spectral source. AFS is different from AES since the latter uses thermal, chemical or electrical mechanism in order to excite electrons into a higher energy level. AAS or AES differs from visible spectrophotometer since the former involves measurement of the absorption/emission of light by vaporized ground state atoms while the latter simply measures the % transmittance and absorbance of a solution as it absorbs certain intensity of light at different wavelengths. The block diagram for visible spectrophotometer, AAS and AES are shown below.

Figure 4.1 Block diagram of visible spectrophotometer.

Figure 4.2 Block diagram of AAS/AES.The added steps in AAS and AES are very vital since these are the reason why AAS and AES can detect concentrations of a specific element. The following diagram shows the scheme in the atomization of a sample subjected to AAS and AES.

Figure 4.3 Scheme for the atomization of sample in Atomic Absorption Spectroscopy.In this exercise, the different components of an AAS system: absorption vs. emission modes was studied. Also, the effects of releasing agents and/or ionization suppressants were determined and standard addition technique and external calibration method for the trace metal analysis were used.II. MATERIALS AND METHODSThe standards and samples were prepared prior to analysis. For the trace analysis of lead, the water sample was prepared by accurately measuring 1.0 L and around 400 mL of it was transferred to a 600-mL beaker. 5.00 mL concentrated HNO3 was then added. The beaker was then placed in a hotplate and the sample was evaporated to almost 100 mL. The remaining amount of water was then added and concentrated by evaporating the sample until the remaining amount was around 100 mL again. Another 5.00 mL concentrated HNO3 was then added and the beaker was then covered with a watchglass. The sample was concentrated to around 50 mL, cooled and filtered. The concentration of lead in the water sample was determined using the external calibration method and standard addition method. For the standard addition method, 0.5 mL from the 1000 ppm Pb2+ stock solution was obtained to prepare 50.0 mL of 10 ppm Pb2+ standard solution. Using this 10 ppm Pb2+ standard solution, 0, 1.0, 2.0, 4.0, 5.0 mL were pipetted into five different 50-mL

volumetric flasks. 5.0 mL of the water sample was then added into each of the volumetric flask and was diluted to mark with deionized water.On the other hand, for the analysis of calcium and potassium, 2.0 g of Bear Band powdered milk was accurately weighed and transferred to an evaporating dish. Three trials were made. The milk sample was placed in a hot plate until almost black. The evaporating dish was then transferred to the furnace at 550℃ for 3-4 hours or until a white/gray residue was observed. The sample was then removed from the furnace and was cooled to room temperature. 2 mL of concentrated HNO3 was then added followed by 10 mL of distilled water. The solution was heated to boiling, cooled and transferred quantitatively while filtering to a 50-mL volumetric flask and was diluted to mark with distilled water. From the 1000 ppm Ca2+ and K+ stock solution, 0.5 mL were obtained from each to prepare 50.0 mL of 50 ppm Ca2+ and 50 ppm K+ standard solutions. Using this 50 ppm Ca2+ and 50 ppm K+ standard solutions, 0, 1.0, 2.0, 3.0, 4.0, 5.0 mL were pipetted into six different 50-mL volumetric flasks. 5.00 mL of 2% La3+ solution was then added to volumetric flasks for calcium analysis while 5.00 mL of 5% Cs solution was supplemented to volumetric flasks for potassium analysis. Each volumetric flask was then diluted to mark using % HNO3. Signals obtained from the working curve were then compared to the signals obtained from the milk sample.All prepared standards and samples were read in AAS. Absorption mode was used for calcium and trace analysis of lead while flame emission mode was used for potassium analysis. Figure 4.1 shows the instrument used in the experiment. Blank readings of each metal analyzed were then subtracted to correct the output absorbance.

Figure 4.4. Atomic Absorption/Emission Spectrometer.

III. RESULTS AND DISCUSSIONAtomic Spectroscopy is a technique for determining the concentration of a particular metal element in a sample. This application took more than 200 years to be well-developed. In 1752, Thomas Melville elaborated on the principles of flame atomic emission spectroscopy. After 100 years, at around 1860, Kirchoff and Bunsen were able to show an analytical relationship between the ground or excited state atoms and the absorption or emission of discrete spectral radiation. Analysis then was done using Bunsen burners and this became the source of problem in terms of the reproducibility of the analytical signal. This problem was solved in 1929 upon the introduction of new burners, nebulizers, gas control devices, and detection systems by Lundegardh. This advancement in spectroscopy was further improved by Sir Alan Walsh and his collaborators in 1955 through the development of hollow cathode lamp serving as the radiation source. L’vov, Greenfield, Fassel, and West, then introduced the non-flame atomizers that minimized the spectral and chemical interferences in the process (Ahuja, 2006).Flame atomic absorption spectroscopy is currently the most widely used of all the atomic spectral methods because of its simplicity, effectiveness and relatively low cost. As shown in the schematic diagram above, solutions are aspirated into the oxidizer gas. Nebulizers, then, pass the liquid aerosol generated into the flame. Upon passing through the flame, the aerosol particles are desolvated, dissociated, and atomized.

The aerosol particle size must be taken into consideration for aerosol droplets that are too large can have insufficient exposure in the flame leading to incomplete atomization aerosol droplets that are too small and can be easily lost through wall collisions. The exposure of the sample in the flame is dependent on the flow rate of the fuel and oxidizer, that’s why optimization should be applied. The flame also has an effect on the degree of atomization. The frequently used flames used in analyses are air-acetylene and nitrous oxide-acetylene. The flame temperature of the gases is 2300oC and 2800OC, respectively. Nitrous oxide-acetylene is recommended in analyses not solely due to higher flame temperature but also to some excited cyanogen radicals that can reduce refractory oxides to atoms. These refractory oxides has the ability to endure flame without atomization and metals such as Al, Ba, Be, Ca, Sc, Si, Ta, Ti, U, V, W, Zr can be in the form of refractory oxides. The air-acetylene, on the other hand, is favored to those elements that have low ionization potentials like Li, Na, K, Rb, and Cs since a flame with higher temperature can ionize the said elements that can result to the reduction of the amount of the metal.The ability of this technique to analyze up to 70 elements is due to the atomizer. The electrons of the atoms in the atomizer can be promoted to higher orbitals for a short amount of time by absorbing a set quantity of energy (i.e. light of a given wavelength). This amount of energy (or wavelength) is specific to a particular electron transition in a particular element, and in general, each wavelength corresponds to only one element. This gives the technique its elemental selectivity. And as the quantity of energy (the power) put into the flame is known, and the quantity remaining at the other side (at the detector) can be measured, it is possible, from Beer-Lambert law, to calculate how many of these transitions took place, and thus get a signal that is proportional to the concentration of the element being measured.The light source used in AAS is usually a discontinuous source since a broad spectrum, produced by a continuous source, is not recommended in AAS analyses. Discontinuous sources (line sources) used today are hollow cathode lamps (HCL) and electrodeless discharge lamps. HCL is manufactured from the element of interest and this lamp is filled with an inert under low pressure. The mechanism of this source is through the production of inert gas discharge that is responsible for the vaporization or sputtering of the element in the cathode. These atoms are then excited via collision with the inert gas ions. Electrodeless discharge lamp, on the other hand, has the same components inside like that of HCL. The discharge is commenced by supplying electrons with a Tesla coil. This type of lamp produces higher intensity atomic spectra than the HCL but the spectral outputs are often unstable.

Like spectrophotometers, AA spectrometers use monochromators and detectors for UV and visible light. The main purpose of the monochromator is to isolate the absorption line from background light due to interferences. Simple dedicated AA instruments often replace the monochromator with a bandpass interference filter. Photomultiplier tubes are the most common detectors for AAS.Also, flame emission spectroscopy or flame photometry has found widespread application in elemental analysis. The instruments are similar to that of flame absorption except that the flame now acts as a radiation source; a hollow- cathode lamp and chopper are therefore unnecessary. The use of flame as atom reservoirs has some disadvantages and one of them is the large consumption of the sample. Due to this, some non-flame atomizers were developed. Electrothermal atomizers such as carbon rods, carbon furnaces and tantalum ribbons are used for AAS and AFS. Atmospheric pressure inductively coupled argon plasmas are used for AES analyses.Quantitative atomic absorption/ emission analyses are based upon the calibration curves involving a plot of absorbance/ emission versus concentration of standards that have been prepared to mimic the solutions of the sample. Standard addition methods are also used extensively in atomic spectroscopy in order to try to compensate for differences between the composition of the standards and the unknowns.Trace metal analysis of lead of water sample via external calibration and standard addition technique was also done in the experiment. Ideally, calibration standards should approximate the composition of the samples to be analyzed with respect to not only the analyte concentration but also the concentrations of the other species in the sample matrix in order to minimize the effects of various components of the sample on the measured absorbance. The standard addition method can take several forms and the multiple addition method is often chosen for photometric or spectrophotometric analyses. This technique involves adding several increments of a standard solution to sample aliquots of the same size. Each solution is then diluted to a fixed volume before measuring its absorbance. When the amount of sample is limited, standard additions can be carried out by successive addition of increments of the standard to a single measured aliquot of the unknown. The measurements are made on the original solution and after each addition of standard analyte. Results are shown below.Table 4.1. Data for trace analysis of lead using external calibration.[Pb2+], ppm Abs

Trial 1 Corrected Trial 2 Corrected0 0.0002 0.0000 0.0014 0.00002 0.0361 0.0359 0.0357 0.03434 0.0655 0.0653 0.0634 0.06206 0.0992 0.0990 0.0971 0.09578 0.1325 0.1323 0.1296 0.128210 0.1597 0.1595 0.1555 0.1541AbsUnknown 0.0764 0.0761Equation of the line y = 0.016x + 0.002 y = 0.0155x + 0.0015R2 0.9989 0.9987Calculated Cx, ppm 4.65 4.812903226Average Cx, ppm 4.731451613Theoretical unknown, ppm 4.5Percent error (%) 5.143369176 %Flow rate: 6.3mL/min

0 2 4 6 8 10 120

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

f(x) = 0.0160057142857143 x + 0.00197142857142857R² = 0.998850486910268

Trial 1

[Pb2+], ppm

Abso

rban

ce

Fig. 4.5. Graph of Trial 1 for trace analysis of lead using external calibration.

0 2 4 6 8 10 120

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

f(x) = 0.0155128571428571 x + 0.00148571428571431R² = 0.998683665202731

Trial 2

[Pb2+], ppm

Abso

rban

ce

Fig. 4.6. Graph of Trial 2 for trace analysis of lead using external calibration.Table 4.2. Data for trace analysis of lead using standard addition method.Vol. of 10 ppm Pb2+, mL AbsTrial 1 Corrected Trial 2 Corrected0 0.0095 0.0000 0.0079 0.00001 0.0129 0.0034 0.0112 0.00332 0.0164 0.0069 0.0145 0.00664 0.0239 0.0144 0.0223 0.01445 0.0261 0.0166 0.0247 0.0168

Equation of the line y = 0.0034x + 7E-05 y = 0.0035x - 8E-05R2 0.9962 0.9968Calculated Cx, ppm 4.117647059 4.571428571Average Cx, ppm 4.344537815

0 1 2 3 4 5 60

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

0.018

f(x) = 0.00341162790697674 x + 7.20930232558162E-05R² = 0.996229575129361

Trial 1

Vol of Pb2+ added, mL

Abso

rban

ce

Fig. 4.7. Graph of Trial 1 for trace analysis of lead using standard addition method.

0 1 2 3 4 5 60

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

0.018

f(x) = 0.00345697674418605 x − 7.67441860465099E-05R² = 0.996817956671431

Trial 2

Vol of Pb2+ added, mL

Abso

rban

ce

Fig. 4.8. Graph of Trial 2 for trace analysis of lead using standard addition method.For calcium, the absorbance of the standards were obtained and plotted against their respective concentrations generating the standard curve. From this curve, interpolation was done to determine the concentration of Ca2+ in the sample. Data were summarized and presented in the tables and figure on the next page.

Table 4.3. Data for Ca analysis.Vol. of 50 ppm Ca2+, mL Abs [Ca2+] Corrected Abs [Ca2+]0 0.0019 0.00001 0.0229 0.02102 0.0474 0.04553 0.0850 0.08314 0.1174 0.11555 0.1513 0.1494Equation of the line y = 0.0305x - 0.0072R2 0.9912Absorbance Calculated CCa, ppmBlank 0 0Trial 1 (m = 2.002 g) 0.0684 619.4020535

Trial 2 (m = 2.023 g) 0.0569 525.1927098Trial 3 (m = 2.013 g) 0.0564 521.0966514Average CCa, ppm 555.2304716Wavelength Ca2+: 422.7 nmDF trial for Ca2+: (50

1 )( 50

10)

0 1 2 3 4 5 60

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

f(x) = 0.0305171428571429 x − 0.00720952380952382R² = 0.991196665873085

Calcium

Vol. of 50 ppm Ca2+, mL

Abso

rban

ce

Fig. 4.9. Linear regression plot for Ca analysis.

On the other hand, potassium analysis was employed using the Atomic Emission technique. The emission of the standards were obtained and plotted against their respective concentrations generating the standard curve. From the curves, interpolation was done to determine the concentration of K+ in the sample. Data were summarized and presented in the table and figure below.Table 4.4. Data for K analysisVol. of 50 ppm K+, mL Emission [K+]0 0.00001 0.06162 0.10603 0.13294 0.15925 0.1954Equation of the line y = 0.037x + 0.0166R2 0.9728Emission Calculated CK, ppmBlank 0 0Trial 1 (m = 2.002 g) 0.1908 235.4054054Trial 2 (m = 2.023 g) 0.1367 162.2972973Trial 3 (m = 2.013 g) 0.1299 153.1081081Average CK, ppm 183.6036036Wavelength K+: 404.0 nmDFtrial for K+: (501

)

0 1 2 3 4 5 60

0.05

0.1

0.15

0.2

0.25

f(x) = 0.0370485714285714 x + 0.0165619047619048R² = 0.972772309749026

Potassium

Vol. of 50 ppm K+, mL

Emiss

ion

Fig. 4.10. Linear regression plot for Ca analysis.The constructed graphs show a direct proportionality between concentration and absorption/emission, which is consistent with Beer-Lambert’s Law. However, the constructed graph is not perfectly linear due to instrumental interferences. This deviation from linearity could be from disproportionate decomposition of molecules at high concentration.It was also experienced in the experiment that there was an over-reading of the Ca absorption and K emission of samples based from the standard solutions. This was troubleshot by diluting the sample. In the case of high concentration of the analyte, other methods can be employed in order to permit accurate determination of the concentration of the analyte. The use of an alternative wavelength having a lower absorptivity as well as the reduction of the path length by rotating the burner hand can be done (Sevostianova).Another factor that caused deviation from the linearity is the presence of interferences. Interferences can be classified into spectral, chemical, and matrix. Spectral interferences occur due to other forms of radiation that overlaps the light source. This is often observed when organic solvents are used and the presence of magnesium in sodium analysis, copper in iron, and nickel in iron analyses. Other spectral interferences are due to some effects that result to the broadening of spectral line. Some of the broadening effects according to Sevostianova are:1. Doppler effect – due to the different components of velocity in the line of observation

2. Lorentz effect – due to the presence of foreign atoms in the environment of emitting or absorbing atoms. The magnitude of the broadening varies with the pressure of the foreign gases and their physical properties.3. Quenching effect - In a low-pressure spectral source, quenching collision can occur in flames as the result of the presence of foreign gas molecules with vibration levels very close to the excited state of the resonance line.4. Self absorption or self-reversal effect - The atoms of the same kind as that emitting radiation will absorb maximum radiation at the center of the line than at the wings, resulting in the change of shape of the line as well as its intensity.Matrix interferences, on the other hand, occur due to the variation in the surface tension and the viscosity of the analyte and the standard solution. Chemical interferences, the most common of all, arise from the ionization of the analyte and the formation of some compounds that do not dissociate in the flame like calcium and strontium phospates, which both leads to a reduction in the signal observed.Treatment of the chemical interferences can be done by manipulating the flame conditions and through the application of certain chemical agents. A low temperature flame can inhibit ionization of the metals and a high temperature flame can further atomize compounds. The other technique is the application of releasing agents, suppressor, and protective agents. A releasing agent like La is a competing ion that reacts with the interfering substance instead of the metal of interest, hence releasing the analyte from interferences. Addition of suppressor like Cs is useful to those elements with low ionization energy like Na. These suppressors, usually with lower ionization energy than the metal of interest, are ionized instead of the metal of interest. On the other hand, protective agents prevent interference by preferentially forming stable but volatile species with the analyte. Application of these techniques leads to a greater amount of sample detection. The application of either a releasing agent or suppressor led to an increase in the concentration of the metal which is consistent with the effect of such agents. These agents act as shields in order to protect the metal of interest from various reaction conditions such as ionization and formation of compounds that are not decomposed in flame.

IV. SAMPLE CALCULATION Trace metal analysis of Lead

Plot absorbance versus volume of aliquotWorking equation:C x=

C sb

V xmWhere:Cx = concentration of unknownb = y-intercept (from the graph of absorbance versus volume of aliquot) Cs = concentration of standard solution = 10 ppmm = slope (from the graph of absorbance versus volume of aliquot)Vx = final volume of unknown = 50 mL or 5x10-3 L Considering Trial 1:From the standard solutions:slope 0.0034intercept 7 x10−5

C x=C sb

V xm

C x=10 ppmx7 x 10−5

0.0034 x5 x 10−3 L=4.117647059 ppm

Average Cx = 4.117647059+4.5714285712

=4.344537815 ppm

Determination of the concentration in the unknown K analysis

Using the equation of the line obtained from Beer’s Law plot: A = abcPlot absorbance (A) vs. concentration (c)y = mx + b

A = ac + b

Where A = absorbancea = absorptivity/slopeb = y-intc = concentrationc =

A-ba

Considering Trial 1:c =

0.1908 -0.01660.037

=4.708108108ppmCalculation of the concentration of K+ in milk sample= 4.708108108ppm x DF (50

1¿ = 235.4054054 ppm

V. CONCLUSIONSFor this experiment, the different components of an AAS system: absorption vs. emission modes was studied. Also, the effects of releasing agents and/or ionization suppressants were determined and standard addition technique and external calibration method for the trace metal analysis were used.Certain considerations must be done in order to have an accurate measurement of the concentration. The aerosol particle size must be taken into consideration for aerosol droplets that are too large can have insufficient exposure in the flame leading to incomplete atomization aerosol droplets that are too small can be easily lost through wall collisions. The flame must be matched with respect to the analyte. A low flame temperature is used for elements with low ionization energy and a high flame temperature is used in elements capable of forming compounds that cannot be decomposed in flame.The atomic absorption spectroscopy still relies on Beer-Lambert’s Law but there are some deviations from the linearity. These deviations from linearity could be from disproportionate decomposition of molecules at high concentration as well as the presence of certain interferences, especially chemical interferences.Chemical interferences can be eliminated through the use of suppressors and releasing agents. Releasing agents are cations that react preferentially with the interferent. The releasing agent that was used in the experiment was

Lanthanum. Suppresants are those that limit the activity of a certain compound. The suppressant used in the experiment was Cesium. These agents act as shields in order to protect the metal of interest from various reaction conditions such as ionization and formation of compounds that are not decomposed in flame. Flame manipulation can also be used to treat interferences.Standard addition technique was used in trace metal analysis. This technique involves adding several increments of a standard solution to sample aliquots of the same size. Each solution is then diluted to a fixed volume before measuring its absorbance. VI. REFERENCESAHUJA S., N. Jespersen. 2006. Comprehensive Analytical Chemistry 47. Elsevier B.V.SEVOSTIANOVA, E. Atomic Absorption Spectroscopy. Retrieved October 1, 2015.SKOOG D.A., D.M. West, F.J. Holler, S.R. Crouch. 2004. Fundamentals of Analytical Chemistry. 8th ed.Brooks/Cole. USA.